Xenopus as a model organism in developmental chemical genetic screens

Chemical genetics is a potentially powerful tool for studying developmental processes in vertebrate systems. We present data showing Xenopus laevis as a model organism in which systematic chemical genetic screens can be carried out. Previous forward chemical genetic screens, including those with developing zebrafish embryos, have demonstrated the nature and value of biological information gained with this approach. We show how amenable Xenopus is to chemical genetics by investigating a series of compounds either with known biochemical effects, or previously identified to give developmental phenotypes, on a range of biological functions, including the development of pigmentation, the heart and the central nervous system in zebrafish. We have found that the compounds give comparable phenotypes when applied to developing Xenopus embryos. We have also studied the penetrance and expressivity of these chemical genetic phenotypes in relation to genetic variation and the developmental window during which the compound is present. Finally, we assess the feasibility and the potential throughput of a screen in this vertebrate species.     

Making the connection: retinal axon guidance in the zebrafish

Genetic screens in zebrafish have identified a large number of mutations that affect neural connectivity in the developing visual system. These mutants define genes essential for accurate retinal axon guidance in the eye and brain and the characterization of these mutants is helping to define the cellular and molecular mechanisms that guide axons in the vertebrate embryo. The combination of zebrafish genetic and embryological approaches promises to greatly increase our understanding of how multiple guidance mechanisms establish the complex neural interconnectivity of the vertebrate brain.     

Probing the enigma: unraveling glial cell biology in invertebrates

Despite their predominance in the nervous system, the precise ways in which glial cells develop and contribute to overall neural function remain poorly defined in any organism. Investigations in simple model organisms have identified remarkable morphological, molecular, and functional similarities between invertebrate and vertebrate glial subtypes. Invertebrates like Drosophila and Caenorhabditis elegans offer an abundance of tools for in vivo genetic manipulation of single cells or whole populations of glia, ease of access to neural tissues throughout development, and the opportunity for forward genetic analysis of fundamental aspects of glial cell biology. These features suggest that invertebrate model systems have high potential for vastly improving the understanding of glial biology. This review highlights recent work in Drosophila and other invertebrates that reveal new insights into basic mechanisms involved in glial development.     

A gain-of-function screen in zebrafish identifies a guanylate cyclase with a role in neuronal degeneration

Manipulation of gene expression is one of the most informative ways to study gene function. Genetic screens have been an informative method to identify genes involved in developmental processes. In the zebrafish, loss-of-function screens have been the primary approach for these studies. We sought to complement loss-of-function screens using an unbiased approach to overexpress genes with a Gal4-UAS based system, similar to the gain-of-function screens in Drosophila. Using MMLV as a mutagenic vector, a cassette containing a UAS promoter was readily inserted in the genome, often at the 5′ end of genes, allowing Gal4-dependent overexpression. We confirmed that genes downstream of the viral insertions were overexpressed in a Gal4-VP16 dependent manner. We further demonstrate that misexpression of one such downstream gene gucy2F, a membrane-bound guanylate cyclase, throughout the nervous system results in multiple defects including a loss of forebrain neurons. This suggests proper control of cGMP production is important in neuronal survival. From this study, we propose that this gain-of-function approach can be applied to large-scale genetic screens in a vertebrate model organism and may reveal previously unknown gene function. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. FJ151012, FJ151013, and FJ151014.     

Chordate origins of the vertebrate central nervous system.

Fine structural, computerized three-dimensional (3D) mapping of cell connectivity in the amphioxus nervous system and comparative molecular genetic studies of amphioxus and tunicates have provided recent insights into the phylogenetic origin of the vertebrate nervous system. The results suggest that several of the genetic mechanisms for establishing and patterning the vertebrate nervous system already operated in the ancestral chordate and that the nerve cord of the proximate invertebrate ancestor of the vertebrates included a diencephalon, midbrain, hindbrain, and spinal cord. In contrast, the telencephalon, a midbrain-hindbrain boundary region with organizer properties, and the definitive neural crest appear to be vertebrate innovations.     

Technology transfer from worms and flies to vertebrates: transposition-based genome manipulations and their future perspectives

To meet the increasing demand of linking sequence information to gene function in vertebrate models, genetic modifications must be introduced and their effects analyzed in an easy, controlled, and scalable manner. In the mouse, only about 10% (estimate) of all genes have been knocked out, despite continuous methodologic improvement and extensive effort. Moreover, a large proportion of inactivated genes exhibit no obvious phenotypic alterations. Thus, in order to facilitate analysis of gene function, new genetic tools and strategies are currently under development in these model organisms. Loss of function and gain of function mutagenesis screens based on transposable elements have numerous advantages because they can be applied in vivo and are therefore phenotype driven, and molecular analysis of the mutations is straightforward. At present, laboratory harnessing of transposable elements is more extensive in invertebrate models, mostly because of their earlier discovery in these organisms. Transposons have already been found to facilitate functional genetics research greatly in lower metazoan models, and have been applied most comprehensively in Drosophila. However, transposon based genetic strategies were recently established in vertebrates, and current progress in this field indicates that transposable elements will indeed serve as indispensable tools in the genetic toolkit for vertebrate models. In this review we provide an overview of transposon based genetic modification techniques used in higher and lower metazoan model organisms, and we highlight some of the important general considerations concerning genetic applications of transposon systems.     

Headwaters of the zebrafish -- emergence of a new model vertebrate

The understanding of vertebrate development has advanced considerably in recent years, primarily due to the study of a few model organisms. The zebrafish, the newest of these models, has risen to prominence because both genetic and experimental embryological methods can be easily applied to this animal. The combination of approaches has proven powerful, yielding insights into the formation and function of individual tissues, organ systems and neural networks, and into human disease mechanisms. Here, we provide a personal perspective on the history of zebrafish research, from the assembly of the first genetic and embryological tools through to sequencing of the genome.     

Screening mosaic F1 females for mutations affecting zebrafish heart induction and patterning

The genetic pathways underlying the induction and anterior-posterior patterning of the heart are poorly understood. The recent emergence of the zebrafish model system now allows a classical genetic approach to such challenging problems in vertebrate development. Two large-scale screens for mutations affecting zebrafish embryonic development have recently been completed; among the hundreds of mutations identified were several that affect specific aspects of cardiac morphogenesis, differentiation, and function. However, very few mutations affecting induction and/or anterior-posterior patterning of the heart were identified. We hypothesize that a directed approach utilizing molecular markers to examine these particular steps of heart development will uncover additional such mutations. To test this hypothesis, we are conducting two parallel screens for mutations that affect either the induction or the anterior-posterior patterning of the zebrafish heart. As an indicator of cardiac induction, we examine expression of nkx2.5, the earliest known marker of precardiac mesoderm; to assess anterior-posterior patterning, we distinguish ventricle from atrium with antibodies that recognize different myosin heavy chain isoforms. In order to expedite the examination of a large number of mutations, we are screening the haploid progeny of mosaic F1 females. In these ongoing screens, we have identified four mutations that affect nkx2.5 expression as well as 21 that disrupt either ventricular or atrial development and thus far have recovered several of these mutations, demonstrating the value of our approach. Future analysis of these and other cardiac mutations will provide further insight into the processes of induction and anterior-posterior patterning of the heart. Dev. Genet. 22:288–299, 1998. © 1998 Wiley-Liss, Inc.     

Ionotropic glutamate receptors in Caenorhabditis elegans

Ionotropic glutamate receptors (iGluRs) are an important class of heteromeric ligand-gated receptor complexes that mediate a large portion of the excitatory neurotransmission in the vertebrate CNS. Since the cloning of the first iGluR subunit in 1989, the study of this receptor family has rapidly developed in mammals and expanded to include the study of conserved glutamate receptors in simpler invertebrate systems, including the fruit fly Drosophila melanogaster and the soil nematode Caenorhabditis elegans. These model organisms have enabled the genetic analysis of glutamate receptors in the context of a simpler nervous system and provided new insights into receptor function and regulation. In this review we will focus on recent studies that have used genetic, behavioral, and electrophysiological techniques to study the function of iGluRs in C. elegans.     

Transposon-mediated mutagenesis of somatic cells in the mouse for cancer gene identification

To successfully treat cancer we will likely need a much more detailed understanding of the genes and pathways meaningfully altered in individual cancer cases. One method for achieving this goal is to derive cancers in model organisms using unbiased forward genetic screens that allow cancer gene candidate discovery. We have developed a method using a “cut-and-paste” DNA transposon system called Sleeping Beauty (SB) to perform forward genetic screens for cancer genes in mice. Although the approach is conceptually similar to the use of replication competent retroviruses for cancer gene identification, the SB system promises to allow such screens in tissues previously not amenable to forward genetic screens such as the gastrointestinal tract, brain, and liver. This article describes the strains useful for SB-based screens for cancer genes in mice and how they are deployed in an experiment.     

Zebrafish Genetic Map with 2000 Microsatellite Markers

The zebrafish is the first vertebrate organism used for large-scale genetic screens seeking genes critical to development. These screens have been quite successful, with more than 1800 recessive mutations discovered that speak to morphogenesis of the vertebrate embryo. The cloning of the mutant genes depends on a dense genetic map. The 2000 markers we present here, using microsatellite (CA) repeats, provides 1.2-cM average resolution. One centimorgan in zebrafish is about 0. 74 megabase, so, for many mutations, these markers are close enough to begin positional cloning by YAC walks.     

Patterning the protochordate neural tube

The complex vertebrate nervous system has evolved from a simpler nervous system such as that seen in present-day protochordates. Through a recent accumulation of gene-expression data, together with fine anatomical studies, we are now able to identify both how the neural tube was patterned when it first evolved and what is truly novel in the vertebrate neural tube. We are entering a new era in the understanding of how the evolution of novel vertebrate structures is linked to genetic evolution.     

Can zebrafish be used as a model to study the neurodevelopmental causes of autism?

The zebrafish has proven to be an excellent model for analyzing issues of vertebrate development. In this review we ask whether the zebrafish is a viable model for analyzing the neurodevelopmental causes of autism. In developing an answer to this question three topics are considered. First, the general attributes of zebrafish as a model are discussed, including low cost maintenance, rapid life cycle and the multitude of techniques available. These techniques include large-scale genetic screens, targeted loss and gain of function methods, and embryological assays. Second, we consider the conservation of zebrafish and mammalian brain development, structure and function. Third, we discuss the impressive use of zebrafish as a model for human disease, and suggest several strategies by which zebrafish could be used to dissect the genetic basis for autism. We conclude that the zebrafish system could be used to make important contributions to understanding autistic disorders.     

Glial cell development and function in the Drosophila visual system

In the developing nervous system, building a functional neuronal network relies on coordinating the formation, specification and survival to diverse neuronal and glial cell subtypes. The establishment of neuronal connections further depends on sequential neuron-neuron and neuron-glia interactions that regulate cell-migration patterns and axon guidance. The visual system of Drosophila has a highly regular, retinotopic organization into reiterated interconnected synaptic circuits. It is therefore an excellent invertebrate model to investigate basic cellular strategies and molecular determinants regulating the different developmental processes that lead to network formation. Studies in the visual system have provided important insights into the mechanisms by which photoreceptor axons connect with their synaptic partners within the optic lobe. In this review, we highlight that this system is also well suited for uncovering general principles that underlie glial cell biology. We describe the glial cell subtypes in the visual system and discuss recent findings about their development and migration. Finally, we outline the pivotal roles of glial cells in mediating neural circuit assembly, boundary formation, neural proliferation and survival, as well as synaptic function.     

The InsP3 receptor: its role in neuronal physiology and neurodegeneration

The InsP3 receptor is a ligand-gated channel that releases Ca2+ from intracellular stores in a variety of cell types, including neurons. Genetic studies from vertebrate and invertebrate model systems suggest that coordinated rhythmic motor functions are most susceptible to changes in Ca2+ release from the InsP3 receptor. In many cases, the InsP3 receptor interacts with other signaling mechanisms that control levels of cytosolic Ca2+, suggesting that the maintenance of Ca2+ homeostasis in normal cells could be controlled by the activity of the InsP3R. In support of this idea, recent studies show that altered InsP3 receptor activity can be partially responsible for Ca2+ dyshomeostasis seen in many neurodegenerative conditions. These observations open new avenues for carrying out genetic and drug screens that target InsP3R function in neurodegenerative conditions.     

Tag team specification of a neural precursor in the Drosophila embryonic central nervous system

The development of vertebrate and invertebrate nervous systems requires the production of thousands to millions of uniquely specified neurons from progenitor neural stem cells. A central question focuses on the elucidation of the developmental mechanisms that function within neural stem cell lineages to impart unique identities to neurons. A recent report⁽¹⁾ details the roles that two genes, pdm-1 and pdm-2, play within an identified neural stem cell lineage in the Drosophila embryonic central nervous system. The results show that pdm-1 and pdm-2 are coexpressed in an identified neural precursor and function redundantly to specify the fate of this cell. As such this report offers an initial view of the genetic programs that create neural diversity.     

Requirement for endoderm and FGF3 in ventral head skeleton formation

The vertebrate head skeleton is derived in part from neural crest cells, which physically interact with head ectoderm, mesoderm and endoderm to shape the pharyngeal arches. The cellular and molecular nature of these interactions is poorly understood, and we explore here the function of endoderm in this process. By genetic ablation and reintroduction of endoderm in zebrafish, we show that it is required for the development of chondrogenic neural crest cells, including their identity, survival and differentiation into arch cartilages. Using a genetic interference approach, we further identify Fgf3 as a critical component of endodermal function that allows the development of posterior arch cartilages. Together, our results reveal for the first time that the endoderm provides differential cues along the anteroposterior axis to control ventral head skeleton development and demonstrate that this function is mediated in part by Fgf3.     

Tol2 transposon-mediated transgenesis in Xenopus tropicalis

The diploid frog Xenopus tropicalis is becoming a powerful developmental genetic model system. Sequencing of the X. tropicalis genome is nearing completion and several labs are embarking on mutagenesis screens. We are interested in developing insertional mutagenesis strategies in X. tropicalis. Transposon-mediated insertional mutagenesis, once used exclusively in plants and invertebrate systems, is now more widely applicable to vertebrates. The first step in developing transposons as tools for mutagenesis is to demonstrate that these mobile elements function efficiently in the target organism. Here, we show that the Medaka fish transposon, Tol2, is able to stably integrate into the X. tropicalis genome and will serve as a powerful tool for insertional mutagenesis strategies in the frog.     

Axon Guidance at the Midline: From Mutants to Mechanisms

How axons in the developing nervous system successfully navigate to their correct targets is a fundamental problem in neurobiology. Understanding the mechanisms that mediate axon guidance will give important insight into how the nervous system is correctly wired during development and may have implications for therapeutic approaches to developmental brain disorders and nerve regeneration. Achieving this understanding will require unraveling the molecular logic that ensures the proper expression and localization of axon guidance cues and receptors, and elucidating the signaling events that regulate the growth cone cytoskeleton in response to guidance receptor activation. Studies of axon guidance at the midline of many experimental systems, from the ventral midline of Drosophila to the vertebrate spinal cord, have led to important mechanistic insights into the complex problem of wiring the nervous system. Here we review recent advances in understanding the regulation of midline axon guidance, with a particular emphasis on the contributions made from molecular genetic studies of invertebrate model systems.